Our long-term goal is to understand how carbohydrate modulates the biological activity of follicle- stimulating hormone (FSH). The human ovary becomes resistant to FSH stimulation with age and circulating FSH concentrations rise, keeping serum estrogen concentrations normal until 2 years before the final menstrual cycle. Around the same time, pituitary FSH changes from predominantly hypo-glycosylated hFSH21 (due to loss of at least one FSH? subunit N-glycan) to a predominantly fully-glycosylated hFSH24, which possesses all 4 N-glycans. Hypo-glycosylated hFSH21 exhibits greater apparent affinity for the FSHR, can occupy more FSHR sites, and associates with FSHR more quickly than fully-glycosylated hFSH24. The overall hypothesis of this proposal is that in the face of a senescing ovary the additional switch from hypo-glycosylated FSH21 to fully-glycosylated FSH24 further compromises fertility and may affect bone. The objectives of this proposal are to study mechanisms for differences in FSHR binding and develop tools to measure FSH glycoform ratios via the following specific aims: 1: Evaluate the roles of FSHR oligomerization and FSH glycosylation on FSH binding site access. Our working hypothesis is that hFSH21 or allosteric modulator binding can change the conformation of FSHRs, thereby increasing the number of available binding sites for FSH24. The resulting greater FSHR occupancy will provoke a correspondingly larger response in gonadal target cells. In aging, hFSH21 abundance decreases, fewer FSHRs are occupied and cellular stimulation is attenuated. 2: Evaluate FSH glycoform abundance as a potential infertility marker. Our working hypothesis is that the hFSH21 to hFSH24 ratio changes during normal physiological events, such as the menstrual cycle and aging. Isolation of both 21k-FSH? and 24k-FSH? subunit glycoforms enhances our ability to elicit glycoform-specific antibodies. Glycoform-specific assays can measure changes in circulating glycoform ratios to define their roles in reproductive physiology. 3. Study the clearance and tissue distribution of FSH21 and FSH24 in the mouse. Our working hypothesis is that FSH21 will be cleared from the circulation more rapidly than FSH24, yet the same mechanism will deliver more FSH21 to target tissues. In bone, where FSH24 is active and FSH21 less active or inactive, FSH24 may preferentially accumulate. The potential translational outcomes of this project include identifying a better diagnostic marker, altered hFSH21/hFSH24 ratio, for reproductive aging diagnosis, and methods to increase the effectiveness of existing pharmaceutical FSH preparations.